Mechanism of human α3β GlyR modulation in inflammatory pain and 2, 6-DTBP interaction

α3β glycine receptor (GlyR) is a subtype of the GlyRs that belongs to the Cys-loop receptor superfamily. It is a target for non-psychoactive pain control drug development due to its high expression in the spinal dorsal horn and indispensable roles in pain sensation. α3β GlyR activity is inhibited by a phosphorylation in the large internal M3/M4 loop of α3 through the prostaglandin E2 (PGE2) pathway, which can be reverted by a small molecule analgesic, 2, 6-DTBP. However, the mechanism of regulation by phosphorylation or 2, 6-DTBP is unknown. Here we show M3/M4 loop compaction through phosphorylation and 2, 6-DTBP binding, which in turn changes the local environment and rearranges ion conduction pore conformation to modulate α3β GlyR activity. We resolved glycine-bound structures of α3β GlyR with and without phosphorylation, as well as in the presence of 2, 6-DTBP and found no change in functional states upon phosphorylation, but transition to an asymmetric super open pore by 2, 6-DTBP binding. Single-molecule Forster resonance energy transfer (smFRET) experiment shows compaction of M3/M4 loop towards the pore upon phosphorylation, and further compaction by 2, 6-DTBP. Our results reveal a localized interaction model where M3/M4 loop modulate GlyR function through physical proximation. This regulation mechanism should inform on pain medication development targeting GlyRs. Our strategy allowed investigation of how post-translational modification of an unstructured loop modulate channel conduction, which we anticipate will be applicable to intrinsically disordered loops ubiquitously found in ion channels.

Chronic pain is a very common problem that affects over 20% individuals in the US and world-wide [22][23][24] .
A derivative of the general anesthetic propofol, 2, 6-di-tert-butylphenol(2, 6-DTBP), that does not bind to γ-Aminobutyric Acid type A (GABA A ) and is non-psychoactive 43 , potentiates α3β GlyR and alleviates hyperalgesia in a mouse model of neuropathic pain 39,44 .The positive modulating effect of 2, 6-DTBP appears to be dependent on the phosphorylation of α3β GlyR 39,41 , and related to an aromatic amino-acid residue near the intracellular opening of the conduction pore 45 .Based on these observations, 2, 6-DTBP seem to represent a promising pain control that speci cally potentiates α3β GlyR without unwanted side effects.However, the working mechanism of 2, 6-DTBP remain mysterious.
The absence of α3β GlyR structure, as well as the lack of understanding in the conformation of the large internal M3/M4 loop, has severely limited our understanding of α3β GlyR regulation in pain conditions, and how new types of pain control molecules may be developed.Here, we report near-atomic resolution structures of the human heteromeric α3β GlyR bound with glycine (α3β-gly GlyR) in both digitonin detergent and nanodiscs, showing minimal differences between them.We also determined structures of phosphorylation mimetic α3S346Eβ-gly GlyR, and its complex with 2, 6-DTBP, α3S346Eβ-gly/2, 6-DTBP GlyR, where we observed asymmetrical pore rearrangements upon phosphorylation and 2, 6-DTBP binding.We further characterized the M3/M4 loop conformation using single molecular uorescence resonance energy transfer (smFRET) method, which revealed changes in loop conformation upon phosphorylation and 2, 6-DTBP binding.Combining structural, electrophysiological and smFRET observations, we propose a mechanism where phosphorylation and 2, 6-DTBP exert functional effects through altering M3/M4 loop conformation that leads to changes in local electrostatics and rearrangements in the TM.This mechanism helps in understanding the regulation of α3β GlyR in in ammatory pain, and possible pharmacological intervention.In addition, it provides a framework for understanding how M3/M4 loop regulate Cys-loop and other related receptors through post-translational modi cation.
Co-expression of 3em and the βem that we reported previously (Extended Data Fig. 1b) 3,5,37 allowed for the assembly of functional 3emβem GlyR, which showed much improved biochemical behavior compared to the wild-type (Extended Data Fig. 1c) while retaining indistinguishable glycine EC 50 (~ 160 µM) and activation Hill slope (~ 3) (Fig. 1b, c, Extended Data Fig. 1f green and light green).S346E had marginal effect on glycine activation, increasing glycine EC 50 of both 3emβem and 3wtβwt GlyR to ~ 180 µM without changing Hill slope (Fig. 1b, c, Extended Data Fig. 1f purple and light purple).
Radii along the pore are very similar to those of 3β-gly structure, suggesting a desensitized functional state.Clearly, phosphorylation at S346 affected TM conformation despite it being situated in the long and unstructured M3/M4 loop.
2, 6-DTBP induced an asymmetric expanded-open pore conformation in the phosphorylation mimic GlyR ( 3S346Eβ-gly/2, 6-DTBP) structure (Fig. 2c, h, i), while maintaining a pseudo-5-fold symmetric ECD (Extended Data Fig. 6d, f).Both the 9' and the − 2' positions dilated asymmetrically, with a minimum pore radius of ~ 3.9 Å at the − 2' position, which is too large for the expected open GlyR pore based on electrophysiology measurements 49,51 .The pore geometry is very similar to previously reported expanded/super open states 5,48,50 .Although 2, 6-DTBP led to dramatic changes in channel conformation, its cryo-EM density cannot be unambiguously identi ed.In addition, the density for M3/M4 loop is missing in all the structures.
Widening of α3: α3 TM interface underlie pore conformational change The structures of 3β-gly, 3S346Eβ-gly and 3S346Eβ-gly/2, 6-DTBP are essentially identical in the ECD, showing pseudo-5-fold symmetry in the glycine-bound conformation with capped C-loops (Extended Data Fig. 6g, h).Differences in the ion conduction pore apparently arose from re-arrangements in the TMs.
Changes of distances in the TM between neighboring α3 subunits contribute to differences in pore conformations.The TM of 3β-gly GlyR, in the desensitized state, showed 5-fold pseudo-symmetry and tight packing (Fig. 3a, g).Non-protein densities were only observed in the conduction pore, and some of which likely correspond to bound ions (Fig. 3d).In contrast, the distance between two α3 subunits (the one next to the α3: β interface) widened in the 3S346Eβ-gly GlyR structure (Fig. 3b).The same α3: α3 interface widened more dramatically in the 3S346Eβ-gly/2, 6-DTBP GlyR (Fig. 3c) structure.A similar increase in inter-α subunit distance in the TM has been observed before in 1β GlyR and found relevant to channel opening 5 .α3 subunits showed similar positive charges in the TM interface (Extended Data Fig. 5j, k) as 1, likely resulting in electrostatic repulsion and promotion of the widening interface.
Since only 1 out of 5 TM interfaces signi cantly widened, 5-fold pseudo-symmetry was broken for both 3S346Eβ-gly and 3S346Eβ-gly/2, 6-DTBP GlyR structures.In addition to the ion conduction pores, extra densities were found in the widened inter-TM gaps (Fig. 3e, f).These densities may represent substances that stabilize the asymmetric TM conformation with larger spaces between one α3: α3 subunit interface (Fig. 3h, i).
α3 M3/M4 loop approximates the pore upon phosphorylation and 2, 6-DTBP binding To understand how phosphorylation at S346, which is ~ 140 Å from the pore if M3/M4 loop is fully extended, affect ion conduction, we used single-molecule Förster resonance energy transfer (smFRET) method to probe the distance between the pore and S346 (Fig. 4a).α3em and βem was engineered to produce α3 FRET β FRET GlyR, with and without S346E mutation, which accept cysteine-reactive chemical dyes only at α3C358 as the acceptor (LD655), and A1-tag-reactive 52,53 dyes between α3S380 and P381 as the donor (LD555, see methods for details).α3C358 is close to the phosphorylation site S346, and α3S380 is at the intracellular terminus of helix M4, next to the pore.α3 FRET β FRET GlyR was immobilized on glass substrate by anchoring the GFP in β subunit M3/M4 loop, mimicking how GlyRs are anchored at post-synaptic densities 32,33,54 .FRET values in this setting (Fig. 4a) re ect on the distance between the phosphorylation site and the ion conduction pore.
smFRET measurements suggest structural exibility in unphosphorylated (α3 FRET β FRET GlyR) M3/M4 loop, which is marginally affect by glycine activation or 2, 6-DTBP binding (Fig. 2b, d and Extended Data Fig. 7a).Unlike systems with more de ned structural states where beautifully discrete FRET states and transitions were observed 55,56 , the FRET signals here appeared more unstable with frequent transitions of non-uniform magnitude, regardless of whether glycine was present (Fig. 4b, top and middle panels, Extended Data Fig. 7a, c).Histograms of average FRET values from multiple detections (apo: n = 184, with glycine: n = 219) shows very similar distributions that decomposes reasonably well to two Gaussians (see methods): one centered around 0.2 FRET value (peak 1) and the other around 0.4 (peak 2), with ~ 75% of population in peak1.Addition of 2, 6-DTBP (n = 247) had small effect in shifting peak 2 to ~ 0.5 FRET value, without affecting population distribution (Extended Data Table 2).
S346E mutation resulted in higher FRET values, which are further increased after application of 2, 6-DTBP (Fig. 4c, e, Extended Data Fig. 7b).FRET values of α3 FRET S346Eβ FRET GlyR, both in apo and in the presence of glycine, can be decomposed into two populations: one centered at ~ 0.3 FRET with 40% counts and anther broader peak centered at ~ 0.5 FRET with ~ 60% (Fig. 4e top and middle panels, Extended Data Table 2).These FRET values are signi cantly larger compared to without S346E mutation, suggesting a more compact M3/M4 loop conformation that brings the phosphorylation site closer to the ion conduction pore.The addition of 2, 6-DTBP dramatically increased FRET, resulting in ~ 67% population with ~ 0.5 FRET and ~ 33% with 0.75 FRET (Fig. 4e bottom panel, Extended Data Table 2), suggesting further compaction of M3/M4 loop towards the pore.That 2, 6-DTBP only increased phosphorylated α3β GlyR activity (S346E, Fig. 1f) coincides with the above measurements where FRET increase is much more evident with the phosphorylation mimic S346E (Fig. 4d, e).

Increased homogeneity in M3/M4 loop distances upon phosphorylation and 2, 6-DTBP binding
To characterize whether the distance between M3/M4 loops from different α3 subunits is modulated by phosphorylation and 2, 6-DTBP binding, we measured FRET e ciencies between C358 of different α3 subunits (Fig. 5a, see methods for details).
After phosphorylation (α3 FRET S346Eβ FRET GlyR), a single component was identi ed centering at ~ 0.5 FRET, also independent of glycine binding (Fig. 5c, e upper and middle panel, apo n = 457, glycine bound n = 433, Extended Data Table 3).Coincidentally, the uctuation of FRET values with respect to time becomes less prominent (Fig. 5c), suggesting a more stable spatial arrangement.3, n = 417), suggesting increased overall compactness and the emergence of a more compact population.After phosphorylation, instead of increasing FRET values, 2, 6-DTBP binding showed a more subtle effect: it further increased the homogeneity of distance distribution, resulting in a narrower peak in histogram (Fig. 5e lower, n = 389).This seemingly inverse correlation between functional effect and loop conformational change hints at the working mechanism of 2, 6-DTBP, and will be discussed later.

Discussion
With engineered α3β GlyR, we resolved structures of the human α3β GlyR before and after phosphorylation (mimic) of the large internal M3/M4 loop, as well as after 2, 6-DTBP potentiation (Fig. 2).Comparison of these structures point to a mechanism where phosphorylation and 2, 6-DTBP regulate ion conduction by changing the conformation of TM in an asymmetric manner (Fig. 3), coinciding with recently proposed asymmetric gating mechanism of heteromeric GlyR 5 .We further show, using smFRET, that phosphorylation leads to compaction of the M3/M4 loop towards the pore, and 2, 6-DTBP binding causes further compaction (Fig. 4), suggesting localized loop-TM interaction underlying TM conformational changes.In addition, we found that 2, 6-DTBP modulates inter-subunit M3/M4 loop distances in a phosphorylation-dependent manner (Fig. 5).
Our ndings suggest an underlying mechanism of how post-translational modi cation in the M3/M4 loop regulates α3β GlyR activity (Fig. 6), which is unlikely through altering the glycine binding pocket 38 since no appreciable difference was identi ed in our structures (Extended Data Figure 6).The M3/M4 loop is randomly positioned below the intracellular pore in non-phosphorylated state (Fig. 6a).Once phosphorylated, the addition of negative charges likely introduces polar interactions within each loop, as well as between loops from different α3 subunits.These interactions lead to a more compact loop conformation with more homogenous distances between loops (Fig. 5e), and reduced distances between the phosphorylation site and the ion conduction pore (Fig. 4e), likely resulting in local accumulation of negative charge and decreased Cl -conduction (Fig. 1e, Fig 6b), explaining how a distal phosphorylation decreases single channel conductance 41 .Changes in loop conformation also affect TM arrangement (Fig. 2f, g and Fig. 3b, e, h), but not su cient to alter functional states and thus has minimal effects in gating 39,41 .The binding of 2, 6-DTBP causes further compaction of the phosphorylated loop toward the ion conduction pore (Fig. 4e, Fig. 6c).Such a dramatic change favors a more expanded TM con guration (Fig. 2 h, i and Fig. 3c, f, i), resulting in altered conduction pore geometry, increasing both unitary conductance and open probability 39,41,45 .We cannot rule out, at present, that the non-protein densities at widened inter-subunit interface (Fig. 3) may be (partly) arising from the M3/M4 loop.
2, 6-DTBP likely elicits function through interaction with M3/M4 loop.Although the density of 2, 6-DTBP cannot be unambiguously identi ed in our map reconstructions, several lines of evidence suggest interaction between 2, 6-DTBP with the M3/M4 loop.Despite having no functional effect when applied to non-phosphorylated GlyR 39,45 (Fig. 1f), 2, 6-DTBP clearly changed the distribution of inter-loop distances (Fig. 5d).This suggests that the 2, 6-DTBP-induced conformational change originates from the M3/M4 loop, which leads to functional effects only when su cient change propagates to the TM.This is consistent with 2, 6-DTBP causing loop compaction toward the conduction pore only for phosphorylated GlyR (Fig. 4d, e).
The of M3/M4 loop regulation mechanism proposed here implies localized interaction within one pentameric GlyR.Only when conformational changes of M3/M4 loop are su cient to induce TM rearrangements, or cause local electrostatic potential change, functional effects arise.Apparently, whether phosphorylation-dependent interactions with unidenti ed players, or between different GlyRs play a role remain unclear.In addition, whether such mechanism is universal among Cys-loop receptors and other ion channels requires further investigation.

Protein expression
Protein α3β and α3S346Eβ was expressed as described before 4,5 .The α3em, α3S346Eem, α3 FRET , α3S346E FRET , βem and β FRET plasmids were transformed into DH10BacY competent cells (Geneva Biotech) to produce bacmids.The bacmids were transfected into Sf9 cells (ATCC, CRL-1711) to generate baculovirus and then recombinant baculovirus titers were measured.Virus was added at MOI (multiplicity of infection) of 2 (at 3βem:1α3em ratio) to HEK293S GnTI -cells (ATCC, CRL-3022) at a density of 2.5×10 6 cells/ml.10 mM sodium butyrate was added, and culture temperature was turned to 30 °C after transduction 12h.Cells were collected after induction 60h by centrifugation at 30,000 g for 20 minutes at 4 °C and stored at −80 °C until further use.Then resin bound with protein were mixed with PreScission protease (1:30 v/v) at RT for 1h to cleave PA tag.The ow through was collected, and resin were washed with another 2 CV buffer B. All proteins were pooled and concentrated to load onto Superose6 increase 10/300 GL column (GE Healthcare) in SEC buffer (20 mM Tris pH8.0, 200 mM NaCl, 2 mM Glycine, 0.05%(w/v) DDM, 0.005% CHS).Reconstitution of α3β GlyR into saposin nanodisc was modi ed from the published protocol 5 .1:30:200 molar ratio of α3β: saposin: brain polar lipids extract (BPE) (Avanti) was used.α3β GlyR protein mixed with BPE at room temperature (RT) for 10 min.Saposin protein was added and the mixture was put at RT for another 2 min.The mixture was diluted with buffer (20 mM Tris pH 8.0, 200 mM NaCl, 2 mM Glycine) and incubate on ice for 30 min.Then bio-beads SM-2 (Bio-Rad) were added to the mixture and rotated overnight at 4 °C.After 12h, old bio-beads were removed and the fresh bio-beads were added for another 10h.The mixture was centrifuged for 30min at 4 ℃ before loading onto Superose 6 increase size exclusion column in SEC buffer (20 mM Tris-HCl pH 8.0, 200 mM NaCl, 2 mM glycine).
Protein (α3β and α3S346Eβ GlyRs) puri cation in digitonin for Cryo-EM data collection Cell lysis and protein solubilization by detergent follow the similar protocol as the protein puri cation for saposin nanodisc reconstitution.Brie y, solubilized membranes were cleared by centrifugation at 40,000g for 30 min.Supernatant was collected and added to PA-tag antibody (NZ-1) resin at RT.The resin was collected and washed with 5 CV buffer B and 5CV buffer C (20 mM Tris pH 8.0, 200 mM NaCl, 2mM MgCl 2 , 1 mM CaCl 2 , 2 mM Glycine, 0.06% (w/v) digitonin (Sigma-Aldrich)).Then, beads were mixed with PreScission protease (1:30 v/v) to cleave PA tag at RT for 1h.The resin was collected to get ow through, then resin was washed another 2 CV buffer C. Flow through and 2CV washed buffer C were pooled and concentrated to load onto Superose6 increase 10/300 GL column in SEC buffer (20 mM Tris-HCl pH8.0, 200 mM NaCl, 2 mM Glycine, 0.06%(w/v) digitonin).Good peak fractions were collected and concentrated to 6 mg/ml for grids freeze.For the sample with 2, 6-DTBP the buffer used throughout the puri cation process contained 500 μM 2, 6-DTBP and another 500 μM 2, 6-DTBP was added to cryo-EM sample for 1h before grid freezing.
Micrographs were collected using a Titan Krios microscope (Thermo Fisher) with a K3 Summit direct electron detector (Gatan) operating at 300 kV using the SerialEM data acquisition software.The GIF-Quantum energy lter was set to a slit width of 20 eV.Images were recorded with the pixel size of 0.415 Å in the super-resolution counting mode.Micrographs were dose-fractioned into 50 frames with a dose rate of 1.4 e -/Å/frame.2-fold binning (0.83 Å pixel size after binning), motion correction and dose weighting of the movie frames were carried out using the Motioncorr2 program 59 .CTF correction was carried out using the CTFFIND 4 program 60 .The following image processing steps were performed in RELION 4 61 .Particles were initially picked using the Laplacian-of-Gaussian blobs and subjected to 2D classi cation to obtain good class-averages.Then good 2D classes were used as template for reference-based auto picking.Resulting particles were extracted with 4-fold binning for a further round of 2D classi cation.Good 2D class-averages were selected and subjected to 3D classi cation using an initial model downloaded from EMDB database (EMD-23148) 3 .For the α3β-gly GlyR in digitonin sample, 1 out of 6 classes in 3D classi cation appeared with good density for the entire channel (Extended Data Fig. 2b).A single density blob for GFP was identi ed for the heteromeric α3β GlyR in digitonin sample.The density arising from GFP fusion on the βem subunit served as ducial marker to differentiate the β subunit from the structurally similar α subunits.A further 3D classi cation into 4 classes with non-binned particles (0.83 Å pixel size) without particle alignment was performed.Partial signal subtraction 62 was carried out to focus on the TMD. 1 indistinguishable good class resulted in a nal of 19,993 particles.After reverting particles to un-subtracted version, CTF re nement, Bayesian polishing in RELION and non-uniform re nement 63 in cryoSPARC 64 , an overall resolution of 3.8 Å was achieved, with local resolutions exceeding 3.5 Å in many regions (Extended Data Fig. 2c, d, e).For the α3β-gly GlyR in nanodisc sample, 1 out of 3 classes in 3D classi cation appeared with good density for the entire channel (Extended Data Fig. 2g).A single density blob for GFP was identi ed for the heteromeric α3β-gly GlyR in nanodisc sample.A further 3D classi cation into 3 classes with non-binned particles (0.83 Å pixel size) without particle alignment was performed.1 indistinguishable good class resulted in a nal of 40,868 particles.After reverting particles to un-subtracted version, CTF re nement, Bayesian polishing in RELION and non-uniform re nement in cryoSPARC, an overall resolution of 3.8 Å was achieved, with local resolutions exceeding 3.5 Å in many regions (Extended Data Fig. 2h, i, j).For the α3S346Eβ-gly GlyR in digitonin, 1 out of 4 classes in 3D classi cation appeared with good density for the entire channel (Extended Data Fig. 3b).A single density blob for GFP was identi ed for the heteromeric α3S346Eβ-gly GlyR sample.A further 3D classi cation into 4 classes with non-binned particles (0.83 Å pixel size) without particle alignment was performed.1 indistinguishable good class resulted in a nal of 9,628 particles.After reverting particles to un-subtracted version, CTF re nement, Bayesian polishing in RELION and non-uniform re nement in cryoSPARC, an overall resolution of 3.7 Å was achieved, with local resolutions exceeding 3.0 Å in many regions (Extended Data Fig. 3c, d, e).
For the α3S346Eβ-gly/2, 6-DTBP GlyR in digitonin sample, 1 out of 6 classes in 3D classi cation appeared with good density for the entire channel (Extended Data Fig. 3g).A single density blob for GFP was identi ed for the heteromeric GlyR α3S346Eβ-gly/2, 6-DTBP GlyR sample.A further 3D classi cation into 4 classes with non-binned particles (0.83 Å pixel size) without particle alignment was performed.1 indistinguishable good class resulted in a nal of 22,755 particles.After reverting particles to unsubtracted version, CTF re nement, Bayesian polishing in RELION and non-uniform re nement in cryoSPARC, an overall resolution of 3.6 Å was achieved, with local resolutions exceeding 2.5 Å in many regions (Extended Data Fig. 3h, i, j).Resolutions were estimated by applying a soft mask around the protein densities with the Fourier Shell Correlation (FCS) 0.143 criterion.Local resolutions were calculated using Resmap 65 .

Model building and re nement
Models of α3β-gly (in digitonin and nanodisc) and α3S346Eβ-gly GlyRs were bulit by tting the structure of heteromeric human α1β desensitized state (PDB ID: 8DN4) 5 into the Cryo-EM density maps of α3β-gly (in digitonin and nanodisc) and α3S346Eβ-gly GlyRs using Chimera 66 and Coot 67 .Model of α3S346Eβgly/2, 6-DTBP GlyR was bulit by tting the structure of heteromeric human α1β expandedopen state (PDB ID: 8DN2) 5 into the Cryo-EM density map of α3S346Eβ-gly/2, 6-DTBP using Chimera 66 and Coot 67 .The atomic model was manually adjusted in Coot.The nal models were re ned with realspace re nement module and validated with comprehensive validation module in PHENIX package 68,69 .Fourier shell correlation (FSC) curves were calculated between re ned atomic model and the work/free half maps as well as the full map to assess the correlation between the model and density map.Statistics of cryo-EM data processing and model re nement are listed in Extended Data Table 1.Pore radii were calculated using the HOLE program 70 .Figures were prepared in UCSF Chimera 66 , ChimeraX 71 , and PyMOL 72 .

Whole cell patch clamp
The glycine EC 50 values were measured on α3β GlyR and α3S346Eβ GlyR expressed in HEK293T cells (ATCC, CRL-3216).Plasmids were transiently transfected using Lipofectamine 3000 reagent (Invitrogen).Total 0.8 μg of DNA was transfected at 1α3:3β ratios for 35 mm dish.Whole-cell recordings were made after 17-24h transfected at 22 ℃.GFP uorescence was used to identify the cells expressing the heteromeric α3β and α3S346Eβ GlyRs.For experiments of PGE 2 modulation GlyR, total 1 μg of plasmid (0.6 μg GlyR at 1α3:3β ratios and 0.4 μg EP 2 ) was transfected for 35 mm dish.Whole-cell recordings were made after 17-24h transfected at 22 ℃.Both in presence of GFP (GlyR) and mCherry (EP 2 ) uorescences were used to identify the cells co-expressing the heteromeric α3β GlyR and EP 2 receptor.PGE 2 (10 μM concentration used) was applied by perfusion system at a rate of 1-2 ml/min.At least 5 times current response evoked by 1 mM glycine was recorded before application of PGE 2 .After application PGE 2 for about 2 minutes, the currents reached steady state.This steady state of inhibition kept another 3 min with PGE 2 application.
Then bath solution without PGE 2 was applied to wash out.
2, 6-DTBP (100 μM concentration used) was also applied by perfusion system at a rate of 1-2 ml/min.After 3 to 5 times current response evoked by 30 μM glycine of baseline recording, 2, 6-DTBP (100 μM) was applied to bath solution for 4-6 min until the currents increase reaching saturation.The increase in current is recorded every 40 seconds.

Protein puri cation and labeling for smFRET
Cell lysis and protein solubilization by detergent follow the protocol as the protein puri cation for saposin nanodisc reconstitution excepting that 20 mM HEPES-NaOH, pH7.4 was used instead of 20 mM Tris-HCl, pH8.0.Peak fractions of protein were collected and concentrated to 1 mg/ml.α FRET β FRET was equally divided into two parts.One part protein was labeled with CoA-LD555 and LD655-MAL.The protocol as described below: 10 μM TCEP was added to protein then incubated for 30 min on ice.α FRET β FRET was labeled rst by incubating protein with LD655-MAL at 1:3 (protein: LD655-MAL) molar ratio at 4 ℃ for overnight in the dark.α FRET β FRET was labeled further by incubating protein with 20 μM AcpS, 10 μM CoA-LD555, 20 mM MgCl 2 , 20 mM HEPES-Na, pH 7.4 at RT for 4h protecting from light.Another part of the protein was labeled with LD555-MAL and LD655-MAL in the dark at 1:3:3 molar ratio (protein: LD555-MAL: LD655-MAL).To remove free dye, the solution with labelled protein was then loaded onto PD-10 desalting column (GE Healthcare) equilibrated in the buffer E (20 mM HEPES-NaOH, pH7.4,200 mM NaCl, 0.03% (w/v) DDM, 0.003% (w/v) CHS), and the resulting ow-through was loaded onto a second desalting column equilibrated in buffer E. The ow through containing pure labeled protein was centrifuged at 18,000g for 1 h at 4 ° C to remove insoluble aggregates.FRET-Labeled α FRET β FRET were aliquoted and frozen at -80 °C, and freshly thawed before the experiments.

Glass preparation for smFRET imaging
The glass slides are cleaned by soaking for 1.5 h at room temperature in piranha solution (≥98% H 2 SO 4 and 30% H 2 O 2 in a 3:1 ratio) in jugs.The procedure is carried out in a hood.The glass slides were sonicated for 3 times for 10s/time (once at the started soaking, once at 45 min and once at the end) and the washed with ddH 2 O for 5 times in jug.Then the treated glass slides are further soaked in 1M KOH for another 30 min and washed for 5 times using running ddH 2 O.During soaking with KOH, the glass slides were sonicated for another 3 times for 1mim/time (once at the started soaking, once at 15 min and once at the end).After washing procedures, the glass slides are drained on air in a vertical position.The soaked glass slides were covered with 25% mPEG-sliane 5k (Sigma-Aldrich) with 1% Biotin mPEG-silane 5k (Sigma-Aldrich) at 90 o C on metal plate covering by Petri dish for 30 min.Finally, the glass slides were washed with running ddH 2 O and then drained on air in a vertical position.Coated glass slides were stored at -20 °C until further use.

TIRF-based single-molecule FRET imaging
For direct immobilization of α FRET β FRET , the imaging surface was rst exposed to 0.2 μM NeutrAvidin (Thermo Fisher Scienti c) and then 50 nM Biotin Anti-GFP antibody (abcam, ab6658) in buffer F (50 mM HEPES-NaOH, pH7.4,150 mM NaCl).The surface was washed and exchanged into imaging buffer (50 mM HEPES-NaOH pH 7.4, 150 mM NaCl, 10 mM MgCl 2 , 0.8% (w/v) glucose).FRETlabeled GlyR variants was diluted to 0.7 nM and bound to a NeutrAvidin/ Biotin anti GFPab-coated glass slide surface for 30 min in imaging buffer with 2 μM 25-nucleotide DNA duplex (IDT) and 10 mg/ml BSA (Jackson Immunoresearch) as surface blocking agents.To measure smFRET in apo state, imaging was performed in imaging buffer.To measure the effect of glycine on smFRET value, imaging was performed in imaging buffer added 2mM glycine.To detect the modulating of 2, 6-DTBP on M3/M4 loop, imaging was performed in imaging buffer added 2 mM glycine and 500 μM 2, 6-DTBP and waiting for 30min before imaging recording.TIRF-based smFRET imaging experiments were performed at 22 °C with a custom-built TIRF microscope.Fluorescence emission from LD555 and LD655 was collected by a 60X, 1.27 NA water immersion objective (Leica), spectrally split in a MultiCam Device (Cairn) and collected with two synchronized Flash 4.0 V3 camera (C13440-20CU, Hamamatsu) with 2x2 pixel binning.SmFRET imaging recordings were performed by exciting with the Gem 560 nm laser (Laser Quantum) laser at 50 mW and acquiring 200 frames per movie at a 200 ms/frame rate in both donor and acceptor channels.

Analysis of TIRF-based single-molecule data
Image movies were analyzed with Cornell SPARTAN version 3.7.0 73following Molecules were detected as local intensity maxima in an image combing with donor and acceptor channels (aligned using the iterative closest points algorithm) averaged over the rst 10 frames and background subtracted with threshold 100.The distances of molecules smaller than 3.5 pixels were excluded from analysis.Traces were extracted from the selected intensity maxima by summing the 9 most intense pixels for each uorescence channel.Selected traces were saved for further analysis if they met the following criteria for experiments recorded with 200 ms (10 ms) time resolution: FRET lifetime > 5, donor acceptor correlation coe cient -1 to 0.5, signal-to-noise >8, #cy3 blinks<4 and remove overlapping traces.Saved races is then manually viewed and selected as all FRET section for further analysis according following criteria: Donor-acceptor uorescence exchange time more than 5s (25 frames); Donor and acceptor uorescence were found to bleach in a single step.Single-molecule traces showing dynamics before photobleaching.More than 180 typically molecules at each condition were manually selected, and FRET values for individual each conduction was accumulated in histograms.Histogram distributions were analyzed with a double Gaussian equation to reveal reoccurring mean FRET values using Origin 2018 software (OriginLab).The correlation results of Gaussian tting analysis were listed on Extended Data Table 2 and 3. FRET histograms showed in results are averaged from the rst 25 frames (total 5s).

Plotting and statistics
Glycine dose-response curves tting was used Origin 2018 software (OriginLab).Plotting for PGE 2 and 2, 6-DTBP modulation GlyRs were carried out by GraphPad Prism (GraphPad Software).Plotting, distribution tting and statistics for all single-molecule data were carried out using Origin 2018 (OriginLab).All errors represent the S.E.M.
The bath solution contained (in mM): 10 HEPES-NaOH pH 7.4, 10 KCl, 125 NaCl, 2 MgCl 2 , 1 CaCl 2 and 10 glucoses.The pipette solution contained (in mM): 10 HEPES-NaOH pH 7.4, 150 KCl, 5 NaCl, 2 MgCl 2 , 1 CaCl 2 and 5 EGTA.The resistance of borosilicate glass pipettes between 2∼7 MΩ.The voltage held at -70 mV and a Digidata 1550B digitizer (Molecular Devices) was connected to an Axopatch 200B ampli er (Molecular Devices) for data acquisition.Analog signals were ltered at 1 kHz and subsequently sampled at 20 kHz and stored on a computer running pClamp 10.5 software.Data analysis was performed by Origin 2018 software (Origin Lab).Hill1 equation was used to t the dose-response data and derive the EC 50 (k) and Hill coe cient (n).For glycine dose response experiment, we t the data using equation , where I is current, I 0 is the basal current (accounting mostly for leak, very close to 0), I max is the maximum current and x is glycine concentration.All start point is xed at 0 during t.Measurements were from 7-11 cells, mean and S.E.M. values were calculated for each data point.

Figure 1 Functional
Figure 1